The metastatic spread of cancer is primarily responsible for the intractable nature of the disease. Interference of cancer metastasis with chemical or biologic agents could perhaps bring about increased survival times in cancer patients. The overall goal of this project is to elucidate steps by which natural cysteine proteinase inhibitors, the cystatins, may act as antimetastatic agents. The long term goal is to target cysteine proteinases as a therapeutic adjunct to traditional treatments for malignant neoplasms. The model to be used in these studies are highly metastatic B 16 mouse melanoma cell lines which have been genetically modified to overproduce cystatin C. In addition, a number of experiments will explore the effect of purified cystatin C on metastatic melanoma cell characteristics such as motility, invasion in vitro, growth and metastasis in vivo. Motility and in vitro invasion will be conducted with modified Boyden chambers. Metastasis studies (in vivo) will be carried out by tail-vein injections of labeled cells or dermal injections of melanoma cells so that tumor growth and metastasis can be measured. Localization studies with antibodies specific for cystatin C and cathepsin B, together with cathepsin B activity measurements will help define the role of cysteine proteinases in metastasis. Specific genetic constructs involving cystatin C will be created to allow marker activities to aid in localization studies. Both conventional fluorescence microscopy and confocal microscopy will be used in localization studies. The specific aims for this project are to determine: l. Does cystatin C overexpression inhibit an early stage of lung colonization? 2. Whether cystatin C inhibits tumor growth, either in clones overexpressing cystatin C or when cystatin C is administered to melanoma tumors in vivo. 3. What effects occur upon exogenous addition of cystatin C to B16 melanoma cells in vitro? 4. Localization of cystatin C by transfection of melanoma cells with gene fusions or modified cystatin in expression vectors. These specific aims will help advance our knowledge of the role of cysteine proteinase inhibitors as anti-metastatic agents. The therapeutic value of this class of inhibitors will be revealed in studies of this nature because the target enzyme and inhibitor are well defined and the B16 melanoma are among the best of characterized metastatic cell lines.